Purpose :
The development of vectors encoding therapeutic genes driven by human photoreceptor-specific promoters is of special interest to increase the efficacy and the safety of gene therapy treatment in photoreceptor degenerations, such as retinitis pigmentosa. At present, however, there is no regulatory approved in vitro model to confirm photoreceptor-specific transgene expression with adeno-associated viral (AAV) vectors. Hence the development of a robust and reproducible in vitro system would facilitate progress towards clinical trials.

Methods :
In this study, we used the retinoblastoma-derived WERI-Rb1 cell line to establish an in vitro model for the expression of a green fluorescent protein (GFP) reporter gene driven by the human rhodopsin kinase (GRK1) or chicken β-actin (CAG) promoter. Recombinant AAV2/8 vectors were prepared using iodixanol gradients (rAAV2/8.CAG.GFP and rAAV2/8.GRK1.GFP). Since several proteins activated during cellular DNA replication and transcription have been reported to interfere with viral DNA replication, we used different enzymatic inhibitors to improve AAV transduction. GFP expression levels were monitored overtime using epifluorescence microscopy.

Results :
Drug toxicity was first assessed using a MTT cell viability assay to find the highest non-toxic concentration. A range of multiplicities of infection (MOI) from 2,000 to 50,000 was tested to give the best transduction efficiency, having used rAAV2/8.CAG.GFP as a positive control. Epifluorescence microscopy analysis revealed that the inhibition of some of these proteins involved in DNA metabolism such as topoisomerases increases the GFP expression under the human photoreceptor-specific promoter GRK1.

Conclusions :
The human GRK1 promoter delivered by the AAV2/8 vector can be assessed in human WERI cells. This study provides a helpful human in vitro model to test the expression of therapeutic genes specifically in photoreceptors using the human GRK1 promoter delivered by the AAV serotype 8 vector, thus making available a reliable and specie-specific preclinical model to validate therapeutic vectors.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.